APPLICANT'S DESCRIPTION: CD40, a member of the TNF-R family, provides critical activation signals to B lymphocytes, including stimulating effector functions, antigen presentation, and memory. Because it plays a central role in immune activation, the CD40 signaling pathway is a target for inhibition and enhancement in experimental clinical therapies for the treatment of autoimmune disease, transplant rejection, and tumor immunotherapy. A clear understanding of the molecular mechanisms of CD40 signaling is thus valuable to both basic and applied immunology. An additional benefit is that information gained about CD40 signaling can potentially provide insights into the signaling mechanisms of other members of the rapidly expanding TNF-R family. This proposal seeks to address unanswered questions about the molecular mechanisms of CD40 signaling. Specific areas investigated are: Aim 1. Determine the roles played by CD40-induced TNF in CD40 effector functions. Recent data show that CD40-induced B cell TNF production makes important contributions to B cell differentiation. Aim 1 will explore the potential roles of TNF in other CD40-mediated B cell functions, and determine the molecular basis for interaction between CD40 and the TNF-R. Aim 2. Characterize how CD40 mediates transcriptional activation, using NFKB-independent regulatory mechanisms. Recent work reveals that CD40-mediated IL-6 production is independent of increases in the transcription factor NF-KB, the focus of most studies of CD40-stimulated transcriptional regulation. The IL6 promoter will thus make an excellent model for study of the additional transcriptional regulatory mechanisms induced by CD40 signaling. Aim 3. Examine molecular mechanisms for synergy between BCR and CD40 signals. BCR ligation markedly enhances many CD40-induced effects, but little is known about the molecular basis for this synergy. Preliminary data provide clues, which have been used to design strategies for answering this question. Aim 4. Analyze the roles played by TRAF molecules in CD40 signaling to B lymphocytes. The TRAF family of cytoplasmic adapter proteins clearly plays a major role in signal transduction by members of the TNF-R family. However, clear and physiologically valid understanding of their biological roles has been complicated by the technical limitations of the model systems used to date to study their functions. It is proposed to use homologous gene targeting to create TRAF/- mature B cell lines, as well as ES cell lines (the latter to be used to reconstitute the hematopoietic system of RAG mice) to study roles of TRAF molecules in mature B cell function, and in immune responses to antigen in the intact animal.